Monoclonal antibody against novel epitopes of foot-and-mouth disease virus protein 3abc and uses thereof

ABSTRACT

This disclosure pertains to isolated antibodies or antigen binding fragments thereof that specifically bind to the 3ABC non-structural protein of Foot-and-Mouth Disease virus (FMDV), wherein the antibodies or antigen binding fragments thereof recognize the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12. Accordingly, this disclosure also pertains to polypeptides having an amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12. Monoclonal antibody Mab 40C8 is also provided. The current disclosure also pertains to methods of detecting FMDV infection in an animal (including assays differentiating infected animals from vaccinated animals (DIVA)) and kits for performing the detection methods. Competitive ELISA kits comprising the antibody or antigen binding fragment thereof and immunoassay plates coated with the polypeptide comprising the amino acid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and/or SEQ ID NO: 12 are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/010,278, filed Jan. 29, 2016, now U.S. Pat. No. 10,035,841.

This invention was made with Government support under HSHQDC-11-X-00189and HSHQDC-09-X-00369 awarded by the United States Department ofHomeland Security Science and Technology (DHS S&T) to the U.S.Department of Agriculture, and under 2007-ST-061-000002-02 andHSHQDC-11-J-00452, awarded by DHS S&T to Texas A&M AgriLife Research.The Government has certain rights in the invention.

The Sequence Listing for this application is labeled “Seq-List.txt”which was created on Dec. 15, 2017 and is 27 KB. The entire contents ofthe sequence listing is incorporated herein by reference in itsentirety.

BACKGROUND

Foot-and-mouth disease (FMD) is a highly contagious viral disease(Foot-and-Mouth Disease Virus (FMDV)) that may affect domestic (e.g.,cattle, swine, sheep, and goats) and wild (e.g., deer, bison, pronghornantelope, and feral swine) cloven-hoofed animals. The disease ischaracterized by fever, vesicular (blister-like) lesions, and subsequenterosions (ulcers) of the surfaces of the mouth, tongue, nostrils,muzzle, feet, and teats. FMD does not typically kill adult livestock,but it does have very detrimental effects on productivity (meat andmilk) and high mortality rates may occur in young animals.

FMD is caused by the FMD virus (FMDV) of the Aphthovirus genus in thePicornaviridae family. There are seven different serotypes of FMDV: O,A, C, Southern African Territories [SAT] 1, SAT 2, SAT 3 and Asia 1.Multiple serotypes co-circulate around the world; six out of the sevenserotypes have been recorded in Africa (O, A, C, SAT 1, SAT 2, SAT 3),while four serotypes (O, A, C, Asia 1) have been documented in theMiddle East and Asia. O serotype is most common, followed by Asia 1. Allserotypes are immunologically distinct but produce clinicallyindistinguishable disease. There is no cross protection betweenserotypes.

Primary infection of ruminants is mainly by the respiratory route,whereas infection of pigs is usually through the oral route. Infectionresults in production of protective serotype specific antibodies againstFMDV structural proteins 5 to 14 days post-infection. Transmission ofFMDV mainly occurs through direct contact between infected andsusceptible animals. Indirect transmission is also possible throughfomites contaminated with secretions and excretions from infectedanimals. FMDV can be found in secretions and excretions such as expiredair, saliva, nasal secretions, milk, urine, feces, and semen fromacutely infected animals. Shedding can occur up to 4 days prior to theonset of clinical signs. Aerosol transmission also occurs, particularlythrough pigs that excrete large amounts of virus through theirrespiratory tract, resulting in infectious aerosols that can be inhaledby other animals in proximity.

FMD is present in about two-thirds of the world and endemic in parts ofAfrica, Asia, the Middle East, and South America. The global economicimpact is colossal due to direct losses associated with reducedproduction efficiency and changes in herd structure, and indirect lossesassociated with cost of control strategies, and loss of internationaltrade status. The estimated annual economic impact of FMD in productionlosses and vaccination cost in endemic regions is estimated between $6.5and $21 billion USD, and $1.5 billion USD in FMD free countries ifoutbreaks occurred, based on reported loss of $20 billion USD during thelast 15 years in countries that were previously considered FMD-free. TheUnited States has been FMD-free since 1929. However, there are manysusceptible animals in the United States, including approximately 94.5million cattle, 67 million swine, and 8.5 million sheep and goats. Anoutbreak of FMD in the U.S. would have a devastating economic impact,due to the loss of international trade, production lost, and costsassociated with depopulation, disposal, and disinfection. Diagnostictesting capabilities to differentiate infected and vaccinated animals(DIVA) are necessary to support emergency vaccination strategies. Tothis end, there is a need for reagents that enables FMD serologicaltesting in the US mainland. The current disclosure provides antibodies,peptides, and kits for detection of FMDV infections and differentiationof FMDV infected animals from FMDV vaccinated animals.

BRIEF SUMMARY

The current disclosure provides isolated antibodies or antigen bindingfragments thereof that specifically bind to the 3ABC non-structuralprotein of Foot-and-Mouth Disease Virus (FMDV), wherein the antibodiesor antigen binding fragments thereof recognize the amino acid sequenceof SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12 as anepitope. Accordingly, the current disclosure provides polypeptidesproviding a novel epitope from FMDV protein 3ABC. The epitope has theamino acid sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5 or SEQ ID NO: 12. The antibodies or antigen binding fragments thereofcan be monoclonal antibodies (Mab), chimeric antibodies, single chainantibodies, single chain fragment variable (scFv) antibodies, orfragment antigen-binding (Fab fragment). In an embodiment, the antibodyis Mab 40C8 as produced by hybridoma which is deposited with theAmerican Type Culture Collection with Designation: PTA-122531.

The current disclosure also provides methods of detecting Foot-and-MouthDisease virus (FMDV) infection in an animal, the method comprisingperforming an assay using the polypeptides (epitope sequences) disclosedherein or antibodies or antigen binding fragments thereof that bind thedisclosed polypeptides on a biological sample obtained from the animal.The assay can be an enzyme-linked immunosorbent assay (ELISA), forexample, sandwich ELISA or competitive ELISA. An infected animal may beinfected naturally (e.g., animals at commercial farms, etc.) orexperimentally (e.g., in laboratories or experimental facility).

The current disclosure also provides kits, for example ELISA kits,comprising the antibody or antigen binding fragment thereof. Theantibodies or antigen binding fragments thereof can be labeled with anenzyme in the ELISA kits. Alternately, the antibodies or antigen bindingfragments thereof can be coated onto immunoassay plates. The kits canfurther comprise an immunoassay plate coated with the polypeptidecomprising the amino acid sequence selected from SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12.

Further, the current disclosure provides antibodies or antigen bindingfragments thereof obtained from an animal that has been immunized with apolypeptide comprising an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12, and wherein theantibodies or antigen binding fragments thereof recognize the amino acidsequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12 asan epitope.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication, withcolor drawing(s), will be provided by the Office upon request andpayment of the necessary fee.

FIG. 1 shows sequence and location of the epitope (SEQ ID NO: 3) boundby the 40C8 antibody within the FMDV protein. The sequence in FIG. 1includes a N-terminal cysteine residue used to conjugate the epitope toa carrier protein and is presented as SEQ ID NO: 6).

FIG. 2 shows a sequence analysis of 331 strains from GenBank whichrevealed up to 20% variability in the 3ABC epitope. The sequences arepresented as SEQ ID NOs: 13-28.

FIG. 3 shows amino acid variability within the 3ABC peptide; thevariability is higher in residues alanine (A), leucine (L) and lysine(K) at positions 4, 7, and 11, respectively.

FIG. 4 shows that Mab 40C8 exhibited positive reactivity to diverse 3ABCpeptide variants. The sequences are presented as SEQ ID NOs: 13-28.

FIG. 5 shows that Mab 40C8 exhibited positive reactivity to the 3ABCrecombinant protein, PET31b3b12X (44.7 kDa; SEQ ID NO: 2), whichcontains sequence variations within the 3B portion of the protein. Left:intact recombinant protein, analyzed by SDS PAGE; Right: specific Mab40C8 detection of recombinant protein using Western blot analysis.

FIG. 6 shows reactivity of Mab 40C8-HRP with 6 major FMDV serotypestested in direct ELISA.

FIGS. 7A and 7B show western blot analysis using Mab 40C8 antibody todetect various serotypes of FMDV. As a control the infected cell-lysateswere also examined using a monoclonal antibody (F19-2) specific to theFMDV 3D polymerase.

FIG. 8 shows schematic of recombinant 3ABC* protein (* indicatesmutation in the protein).

FIG. 9 shows schematic representation of entire plasmid for theexpression of FMDV 01 Campos mutant 3ABC* protein (SEQ ID NO: 29 and SEQID NO: 30).

FIG. 10 shows SDS PAGE gel of recombinant 3ABC* protein. L: molecularweight markers; A: lot A; B: lot B.

FIG. 11 shows detection of antibodies by the 3ABC ELISA followingInfection of cattle with one of four serotypes of FMDV (n=4animals/serotype; data averaged).

FIG. 12 shows detection of antibodies to the FMDV 3ABC non-structuralproteins in cattle vaccinated with Ad-A24.

FIG. 13 shows percent inhibition (% I) distribution of 486 bovine FMDVnegative and 139 FMDV positive samples. The vertical line denotes the45% I cut-off; Dx Se denotes diagnostic sensitivity; Dx Sp denotesspecificity.

FIG. 14 shows percent inhibition (% I) distribution of 491 bovinenegative samples. The vertical line denotes the 45% I cut-off; Dx Spdenotes diagnostic specificity.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1: Sequence of 3ABC protein from FMDV serotype O.

SEQ ID NO: 2: Amino acid sequence for the 3ABC recombinant proteindesignated PET31b3b12X.

SEQ ID NO: 3: Sequence of the epitope for Mab 40C8 binding. (Sequence:GPYAGPLERQKPLK).

SEQ ID NO: 4: Sequence of the minimal epitope for Mab 40C8 binding.(Sequence: GPLERQ).

SEQ ID NOs: 5 and 12: Alternate sequence of the minimal epitopes for MAB40C8 binding, where X is any amino acid. (Sequence: G X₁X₂ERQ;GPYAGX₁X₂ERQKPLK).

SEQ ID NO: 6: FMDV peptide for generation of Mab 40C8 antibody withamino terminal cysteine added. (Sequence: CGPYAGPLERQKPLK).

SEQ ID NO: 7: Forward PCR primer for overlap extension PCR of 3ABC cDNA.(Sequence: CAATTCCTTCCCAAAAATCT).

SEQ ID NO: 8: Reverse PCR primer for overlap extension PCR of 3ABC cDNA.(Sequence: GTGGTGTGGTTCGGGGTCCAA).

SEQ ID NO: 9: Nucleotide sequence of plasmid pET30c O1C 3ABC* (3Cmutation at nucleotide 6381: T to C, Cysteine to Arginine).

SEQ ID NO: 10: Nucleotide sequence encoding O1C 3ABC* (3C mutant atnucleotide 1239; T to C, Cysteine to Arginine).

SEQ ID NO: 11: Amino acid sequence corresponding to the serotype O FMDV3ABC* mutant protein containing a His6 tag for expression andpurification from Escherichia coli (3C mutant at amino acid 163:Cysteine to Arginine).

SEQ ID NOs: 13-28: Peptide sequences disclosed in FIGS. 2 and 4.

DETAILED DISCLOSURE Overview

We have developed a monoclonal antibody (40C8) specific for the FMDVnonstructural protein (NSP) 3ABC polypeptide and have demonstrated broadreactivity of this antibody against bovine sera from all seven FMDVserotypes. This monoclonal antibody may be used for FMDV serologicaldiagnostics (including differentiation between infected and vaccinatedanimals (DIVA) capability) and attenuated FMD vaccine production qualitycontrol testing.

Foot-and-mouth disease (FMD) serological testing in the U.S. iscurrently performed only at the USDA Animal and Plant Health InspectionService (APHIS) Foreign Animal Disease Diagnostic Laboratory (FADDL) atPlum Island Animal Disease Center (PIADC) under an experimental researchand evaluation permit using an ELISA kit. The current disclosureprovides antibodies, peptides, and kits for detection of FMDV infectionsand differentiation of FMDV infected animals from FMDV vaccinatedanimals.

Disclosure

ATCC information: The hybridoma cell line which can be used to produceMab 40C8 was deposited with American Type Culture Collection (ATCC),P.O. Box 1549, Manassas, Va. 20108, on Sep. 29, 2015 (ATCC DesignationPTA-122531). The subject hybridoma cell line has been deposited underconditions that assure that access to the cell line will be availableduring the pendency of this patent application to one determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37C.F.R. 1.14 and 35 U.S.C. § 122. This deposit will be available asrequired by foreign patent laws in countries wherein counterparts of thesubject application, or its progeny, are filed. However, it should beunderstood that the availability of a deposit does not constitute alicense to practice the subject invention in derogation of patent rightsgranted by governmental action.

The current disclosure provides a novel epitope of the 3ABC protein fromFMDV, for example, an epitope having the amino acid sequence of SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12 (or any polypeptidedisclosed in FIG. 4), and antibodies and antigen binding fragmentsthereof that specifically recognize one of these epitopes. Thisdisclosure also describes methods, techniques, approaches, kits for usein conjunction with the present disclosure.

The current disclosure also provides methods of using the antibodies orantigen binding fragments thereof and the novel epitopes for detectionfor FMDV infection in animals and for distinguishing FMDV infectedanimals from animals vaccinated against FMDV infection. The methods ofthe current disclosure provide improved sensitivity and specificity overthe existing methods of detection of FMDV in animals and reduces thetime necessary to identify animals having a positive serotype for FMDV.

Accordingly, the current disclosure provides antibodies and antigenbinding fragments thereof that specifically binds to the 3ABCnon-structural protein of FMDV, wherein the antibodies or antigenbinding fragments thereof recognize the amino acid sequence of SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 12 (or any polypeptidedisclosed in FIG. 4) as an epitope. In some embodiments, antibodies bindto the same epitope as the monoclonal antibody produced by the hybridoma40C8. Such antibodies can be identified using any one of a variety ofimmunological screening assays in which antibody competition can beassessed. In an embodiment, the antibody is a monoclonal antibody.Additionally, the antibodies can be polyclonal antibodies. In furtherembodiments, the antibody or antigen binding fragments thereof can bechimeric antibodies, single chain antibodies, scFv antibodies, or Fabfragments.

Thus, one embodiment provides a monoclonal antibody, Mab 40C8, asproduced by hybridoma cell line deposited with the American Type CultureCollection with Designation: PTA-122531.

A further embodiment provides antibodies or antigen binding fragmentsthereof obtained from an animal that has been immunized with apolypeptide comprising an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO: 5 or SEQ ID NO: 12 (or any polypeptide disclosed in FIG.4), and wherein the antibodies or antigen binding fragments thereofrecognize the amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5 or SEQ ID NO: 12 (or the immunizing polypeptide from FIG. 4) as anepitope. These antibodies can also be monoclonal antibodies orpolyclonal antibodies.

The antibodies and antigen binding fragments thereof can be used in anassay to detect the presence or absence in a sample of 3ABC protein fromFMDV, for example by a Western blot analysis or a sandwich ELISA; todetect the presence in a sample of antibodies against 3ABC protein fromFMDV, for example, by a competitive ELISA; or to detect the presence ofFMDV infection in an animal, for example, by a western blot analysis oran ELISA. The antibodies and antigen binding fragments thereof can alsobe used to distinguish the animals infected with FMDV from the animalsvaccinated against FMDV. Accordingly, the current disclosure providesmethods of detecting the presence of 3ABC protein, or antibodies against3ABC proteins and thus detect the presence of FMDV infection in ananimal.

The detection of the presence of 3ABC protein, or the antibodies against3ABC proteins can be facilitated by conjugating/coupling the antibodiesand antigen binding fragments thereof to appropriate labels.Non-limiting examples of labels that can be conjugated to the antibodiesor antigen binding fragments thereof as disclosed herein include anenzyme, a radioisotope, a fluorescent label, or a bioluminescent label.Additional embodiments of labels that can be conjugated/coupled to theantibodies and antigen binding fragments thereof of the currentdisclosure and various methods of detecting the labels are recognized.

Various assays for detection of 3ABC proteins can be used to detect FMDVinfection in an animal using the antibodies or antigen binding fragmentsthereof of the current disclosure. Non-limiting examples of proteindetection assays include Western blot analysis, ELISA,immunohistochemistry, or immunoprecipitation. Additional examples ofassays utilizing antibodies or antigen binding fragments thereof todetect the presence of specific proteins or specific antibodies insamples are recognized.

Assays disclosed herein can be used to detect the 3ABC protein, orantibodies against 3ABC proteins can be performed on a biological sampleobtained from an animal. In certain embodiments, the biological sampleobtained from the animal contains the 3ABC proteins derived from FMDV orthe antibodies produced in the body of the animal against 3ABC proteins.In embodiments the biological sample is a body-fluid sample or a tissuesample. It should be appreciated that the assays may be used as part ofan approach to determine the absence of the 3 ABC protein in someinstances.

This disclosure also provides methods of screening a population ofanimals for FMDV infection, an exemplary method includes:

a) obtaining a biological sample from each animal from the population ofanimals,

b) conducting an assay comprising contacting said biological sample withan 3ABC polypeptide or a polypeptide comprising SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO: 4, SEQ ID NO: 5, any polypeptide disclosed in FIG. 4 orSEQ ID NO: 12, said polypeptide being optionally bound to a substrate,using an antibody or an antigen binding fragment thereof to distinguishFMDV infected animals from animals vaccinated against FMDV infection,wherein the antibody or the antigen binding fragment thereof recognizesthe amino acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, anypolypeptide disclosed in FIG. 4 or SEQ ID NO: 12 as an epitope, and

c) identifying individual animals from the population of animals as FMDVinfected animals or FMDV vaccinated animals. In certain embodiments, theassay is performed using the kit according to the current disclosure. Ina further embodiment, the animal screened for FMDV infection is at leastone of cattle, buffalo, water buffalo, sheep, goat, antelope, deer,bison, elephant, llama, or alpaca. The sample obtained from the animalcan be any biological sample described above. Additional embodimentsprovide for the optional quarantine or slaughter of infected animals.

Non-limiting examples of biological samples (e.g., body fluid samples)include aqueous humor, vitreous humor, blood serum, blood plasma,cerebrospinal fluid, endolymph, perilymph, exudates, lymph, mucus,pericardial fluid, pleural fluid, synovial fluid, milk, or oral fluids.Non-limiting examples of tissue samples include brain, eyes, pinealgland, pituitary gland, thyroid gland, parathyroid glands, thorax,heart, lungs, esophagus, thymus gland, pleura, adrenal glands, appendix,gall bladder, urinary bladder, large intestine, small intestine,kidneys, liver, pancrease, spleen, stoma, prostate gland, testes,ovaries, or uterus. The tissue samples can be appropriately processed toproduce a sample suitable for performing the assay for detection of the3ABC protein, or antibodies against 3ABC proteins. It is to be apparentthat the foregoing method can also be used to identify animals that havebeen innoculated for protection against FMD as is understandable to aperson of ordinary skill.

The current disclosure further provides kits containing the antibodiesor antigen binding fragments thereof. A kit, for example, is used todetect the presence in a sample of 3ABC protein, to detect the presencein a sample of antibodies against 3ABC protein from FMDV, or to detectthe presence of FMDV infection in an animal. Using the antibodies orantigen binding fragments described herein, a person of ordinary skillin the art can design various assays for different purposes inaccordance with this disclosure, for example, to detect the presence of3ABC protein, to detect the presence of antibodies against 3ABC protein,or to detect the presence of FMDV infection in an animal. Antibodiesthat bind to an epitope defined by any one of SEQ ID NO: 3, 4, 5 or 12are also expected to bind to proteins containing this epitope (e.g., the3B, 3AB, 3ABC or the recombinant protein of SEQ ID NO: 2).

The kits in accordance with this disclosure can comprise reagents forconducting various assays. The reagents provided in a kit depend on thepurpose and design of the assay to be performed. For example, a kitdesigned for a western blot analysis contains a labeled secondaryantibody and additional reagents for visualizing the label. Variousembodiments of western blot analyses are contemplated.

Kits for performing ELISA are also disclosed. The kits can be designedto perform different types of ELISA, for example, a sandwich ELISA or acompetitive ELISA. A person of ordinary skill in the art knows variousreagents and tools required for conducting each of the different typesof ELISAs and accordingly, the current disclosure provides kitscontaining appropriate reagents and tools for performing these ELISAs.

For example, a kit for conducting a sandwich ELISA can contain animmunoassay plate coated with the antibodies or antigen bindingfragments thereof disclosed herein. The sandwich ELISA kit can furthercontain 3ABC antibodies directed to different epitopes than the epitopesof the current disclosure. These different antibodies can be conjugatedto an enzyme. The sandwich ELISA kit can further contain reagents forcarrying out and visualizing the enzymatic reaction catalyzed by theconjugated enzyme.

A person of ordinary skill in the art with this disclosure can designvarious embodiments of a sandwich ELISA kit using the antibodies orantigen binding fragments thereof disclosed herein. For example, insteadof coating the immunoassay plate with the antibodies or antigen bindingfragments thereof, a kit contains immunoassay plate coated withantibodies against 3ABC epitopes different from the epitopes disclosedherein and enzymatically labeled antibodies or antigen binding fragmentsthereof of the current disclosure. Alternately, enzyme linked secondaryantibodies can also be provided. Additional designs of ELISAs can beenvisioned based on the present disclosure.

A further embodiment of this disclosure provides a kit for performingcELISA for detection of antibodies against 3ABC protein in a sample, forexample a biological sample obtained from an animal. The USDA andDepartment of Homeland Security (DHS) Science and Technology researchteams at Plum Island Animal Disease Center (PIADC) have developed thefirst bioengineered vaccine against FMDV that can be produced in theU.S. The cELISA kit of the current disclosure provides a companiondiagnostic tool for a U.S.-based FMD vaccination program that utilizesFMD vaccines lacking a full-length 3ABC protein. Therefore, the kit canbe used to detect FMDV infection in animals and can also differentiateinfected animals from vaccinated animals, animals vaccinated forimmunity to FMDV. For example, animals vaccinated with FMD vaccineslacking a full-length 3ABC protein or FMD vaccines that do not code forthe FMDV 3ABC protein lack antibodies that bind to the 3ABC polypeptideor the epitope bound by the antibodies disclosed herein. Thus, suchanimals should not have antibodies that bind to a polypeptide disclosedherein and such animals should not have antibodies that compete with thedisclosed antibodies (e.g., such animals should have no antibodies thatcompete with the 40C8 monoclonal antibody (or antibodies that bind tothe same epitope as the 40C8 antibody) for binding to a peptide asdisclosed herein or the 3ABC polypeptide).

The cELISA kit described herein can provide a fast FMD serological assaywhich provides results in hours rather than days, as well as superiorspecificity and sensitivity compared to a commercial product. The cELISAkit of the current disclosure also provides a differentiation betweeninfected and vaccinated animals (DIVA) test for the adenovirus serotype5 FMD vaccine (and future vaccines that lack FMDV 3ABC immunogenicproteins. Certain aspects of this disclosure provide polypeptides usefulfor making antibodies or in competitive immunoassays. With respect tothe amino acid sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 12, X₁and X₂ can be any amino acid (as set forth in Table 1), provided that ifX₁ is proline, then X₂ cannot be leucine and if X₂ is leucine, then X₁cannot be proline (e.g., SEQ ID NO: 12 specifically excludes the aminoacid sequence of SEQ ID NO: 3 as a possible sequence). Anotherembodiment provides, with respect to the amino acid sequences set forthin SEQ ID NO: 5 and SEQ ID NO: 12, that X₁ and X₂ can be any amino acid(as set forth in Table 1), provided that if X₁ is proline, then X₂cannot be methionine. Other non-limiting examples of polypeptides areprovided in FIG. 4). In some other embodiments, polypeptides in which acysteine (C) is found at the amino terminus of the polypeptide are alsoprovided (e.g., SEQ ID NO: 6). Polypeptides containing a cysteineresidue at the amino terminus of the polypeptide are suitable forcovalent attachment to a carrier protein via another cysteine residue orvarious linkers.

TABLE 1 20 amino acids and single letter codes (SLC) Amino Acid SLCIsoleucine I Leucine L Valine V Phenylalanine F Methionine M Cysteine CAlanine A Glycine G Proline P Threonine T Serine S Tyrosine Y TryptophanW Glutamine Q Asparagine N Histidine H Glutamic acid E Aspartic acid DLysine K Arginine R

In another aspect, immunoassays using the disclosed polypeptides orantibodies are provided. In embodiments in accordance with this aspect,antibodies that bind to an epitope comprising SEQ ID NO: 3, 4, 5 or 12(or the polypeptides disclosed in FIG. 4) or a polypeptide selected fromSEQ ID NO: 2, 3, 4, 5 or 12 (or polypeptides disclosed in FIG. 4) arebound to a substrate. The term “bound” refers to both covalent andnon-covalent attachment of an antibody or polypeptide to a substrate.Thus, antibodies or polypeptides can be covalently bound to thesubstrate via a linker physically attached to a substrate ornon-covalently bound to a substrate (e.g., adsorbed to a substratesurface, for example, a polystyrene surface).

In various embodiments, the substrate is one or more tubes, cylinders,beads, discs, silicon chips, microplates, polyvinylidene difluoride(PVDF) membrane, nitrocellulose membrane, nylon membrane, porousmembranes, non-porous membranes, plastic, polymer, silicon, polymericpins, a plurality of microtiter wells, or combinations thereof. Thecomposition of the substrate can also be varied. Substrates(alternatively referred to as a support) can comprise glass,cellulose-based materials, thermoplastic polymers, such as polyethylene,polypropylene, or polyester, sintered structures composed of particulatematerials (e.g., glass or various thermoplastic polymers), or castmembrane film composed of nitrocellulose, nylon, polysulfone, or thelike. Thus, the substrate may be any surface or support upon which anantibody or a polypeptide selected from those disclosed in FIG. 4 or SEQID NOs: 2, 3, 4, 5, 6 and/or 12 can be immobilized, including one ormore of a solid support (e.g., glass such as a glass slide or a coatedplate, silica, plastic or derivatized plastic, paramagnetic ornon-magnetic metal), a semi-solid support (e.g., a polymeric material, agel, agarose, or other matrix), and/or a porous support (e.g., a filter,a nylon or nitrocellulose membrane or other membrane). In someembodiments, synthetic polymers is used as a substrate, including, e.g.,polystyrene, polypropylene, polyglycidylmethacrylate, aminated orcarboxylated polystyrenes, polyacrylamides, polyamides,polyvinylchlorides, and the like. In preferred embodiments, thesubstrate comprises a microtiter immunoassay plate or other surfacesuitable for use in an ELISA.

In embodiments, polypeptides have additional material covalently linkedto either or both ends of the polypeptide (e.g., additional amino acidsto the amino acid sequence of interest), provided that, in the case ofSEQ ID NOs: 3, 4, 5, 6 or 12 (or the polypeptides disclosed in FIG. 4),the additional amino acids do not provide the sequence of the FMDV 3ABCor any other naturally occurring polypeptide containing SEQ ID NO: 3, 4,5 or 12 (or sequence as disclosed in FIG. 4). Covalent linkage ofadditional amino acids to either or both ends of the polypeptidedisclosed herein results in a combined amino acid sequence that is notnaturally occurring, e.g. an unnatural amino acid sequence that is notfound in nature. Polypeptides include a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO: 4, 5, 6 or 12(or any polypeptide disclosed in FIG. 4) coupled to a carrier protein(e.g., a carrier such as an albumin (e.g., bovine serum albumin),keyhole limpet hemocyanin, ovalbumin). Such coupling can be a covalentlinkage. These peptides are rendered immunogenic by coupling them to animmunogenic carrier by the following procedure.

An immunizing agent is constructed by covalently conjugating apolypeptide selected from the polypeptides disclosed in FIG. 4, SEQ IDNOs: 4, 5, 6 and/or 12 to an immunogenic carrier protein, preferably bymeans of a crosslinker, such as a glutaraldehyde moiety or other knownlinkers. Immunogenic carriers include compounds to which any one of thepeptides disclosed herein attached so as to render the peptideimmunogenic. Non-limiting examples of such carriers include proteinssuch as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA),ovalbumin (OVA), tetanus toxoid and human thyroglobulin. One function ofthe crosslinker is to introduce into the immunizing agent a spacer ofsufficient size to prevent the carrier protein from masking thepolypeptide. Suitable crosslinking agents are known in the art and arecommercially available. The immunizing agent is then administered to asubject, such as a goat, sheep, rabbit, mouse, rat, guinea pig, pig,cow, buffalo or other appropriate non-human mammal in an amountsufficient to raise an immune response to the immunizing agent,specifically the polypeptide of SEQ ID NO: 2, 4, 5, 6 or 12 or any ofthe polypeptides disclosed in FIG. 4. The immunizing agent can beadministered in combination with an adjuvant, such as Freund's Completeadjuvant, Freund's Incomplete adjuvant, aluminum salts, oil basedadjuvants, saponins, chemically synthesized adjuvants, or otheradjuvants.

Another embodiment provides for the use of the disclosed peptides forFMDV antigen or antibody detection. For example, the peptide can be usedto coat an ELISA plate in a competitive or indirect ELISA format todetect FMDV antibody from diverse samples (e.g., natural or experimentalinfections). Alternatively, the peptide can be used in a liquid phasecompetitive ELISA for detection of FMDV antigen. In this example, anELISA plate is coated with the 3ABC monoclonal antibody and a liquidsample containing the FMDV 3ABC antigen is then added and allowed tocompete with a constant concentration of 3ABC peptide. Thus, one candetect 3ABC contaminants in vaccine preparations.

An example cELISA kit includes:

-   -   a) the antibodies or antigen binding fragments thereof        optionally labeled with an enzyme or other label,    -   b) an immunoassay plate or other substrate coated with a        polypeptide comprising an amino acid sequence selected from        those disclosed in FIG. 4, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID        NO: 5, SEQ ID NO: 6 and/or SEQ ID NO: 12,    -   c) if the antibody or antigen binding fragment thereof is not        labeled with an enzyme or other label, an antibody against the        antibody or antigen binding fragment thereof conjugated to an        enzyme or label, and    -   d) reagents and tools for conducting the cELISA assay.

The antibody or antigen binding fragment thereof of the currentdisclosure provided in the kit can be chimeric antibodies, single chainantibodies, scFv antibodies, or Fab fragments. In one embodiment, thekit contains Mab 40C8 antibody as produced by hybridoma which isdeposited with the American Type Culture Collection with Designation:PTA-122531.

The immunoassay plate can be coated with a polypeptide consistingessentially of the amino acid sequence selected from those disclosed inFIG. 4, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and/orSEQ ID NO: 12 (or any combination thereof). Non-limiting examples ofpolypeptides that can be conjugated to the immunoassay plate includepeptides consisting of those disclosed in FIG. 4, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and/or SEQ ID NO: 12; 3ABC protein; or3ABC protein carrying a mutation rendering it protease resistant(described in detail in the Materials and Methods section and theExamples below). Any peptide containing the epitopes for the antibodiesor antigen binding fragments thereof disclosed herein can be used tocoat the immunoassay plate and such embodiments are within the purviewthis disclosure.

The current disclosure also provides the following non-limitingembodiments:

1. An isolated antibody or antigen binding fragment thereof thatspecifically binds to 3ABC non-structural protein of Foot-and-MouthDisease virus (FMDV), wherein the antibody or antigen binding fragmentthereof specifically binds an epitope consisting essentially of SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12.

2. The antibody or antigen binding fragment thereof of embodiment 1 thatspecifically binds the amino acid sequence of SEQ ID NO: 3, SEQ ID NO:6, or SEQ ID NO: 12 as the epitope.

3. The antibody or antigen binding fragment thereof of embodiment 1 thatspecifically binds the amino acid sequence of SEQ ID NO: 4 as theepitope.

4. The antibody or antigen binding fragment thereof of embodiment 1 thatspecifically binds the amino acid sequence of SEQ ID NO: 5 as theepitope.

5. The antibody of embodiment 1, wherein the antibody is a monoclonalantibody.

6. The antibody of embodiment 1, wherein the antibody is a polyclonalantibody.

7. The antibody or antigen binding fragment thereof of embodiment 1,wherein the antibody is selected from a chimeric antibody, a singlechain antibody, a single chain fragment variable (scFv) antibody, or afragment antigen-binding (Fab fragment).

8. The antibody of embodiment 1, wherein the antibody is Mab 40C8 asproduced by hybridoma which is deposited with the American Type CultureCollection with Designation: PTA-122531.

9. The antibody or antigen binding fragment thereof of embodiment 1,wherein the antibody or antigen binding fragment thereof is conjugatedto a label.

10. The antibody of embodiment 9, wherein the label is selected from anenzyme label, a radioisotope, a fluorescent label, or a bioluminescentlabel.

11. A method of detecting FMDV infection in an animal, the methodcomprising contacting a sample from an animal with an antibody orantigen binding fragment thereof of that specifically binds to the 3ABCnon-structural protein of FMDV, wherein the antibody or antigen bindingfragment thereof specifically binds the amino acid sequence of SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 SEQ ID NO: 6, or SEQ IDNO: 12.

12. The method of embodiment 11, wherein the sample obtained from theanimal is a body-fluid sample or a tissue sample.

13. The method of embodiment 12, wherein the body-fluid sample isaqueous humor, vitreous humor, blood serum, blood plasma, cerebrospinalfluid, endolymph, perilymph, exudates, lymph, mucus, pericardial fluid,pleural fluid, synovial fluid, milk, or oral fluids.

14. The method of embodiment 12, wherein the tissue sample is brain,eyes, pineal gland, pituitary gland, thyroid gland, parathyroid glands,thorax, heart, lungs, esophagus, thymus gland, pleura, adrenal glands,appendix, gall bladder, urinary bladder, large intestine, smallintestine, kidneys, liver, pancreas, spleen, stoma, prostate gland,testes, ovaries, or uterus.

15. The method of embodiment 11 or 12, wherein the assay is a Westernblot analysis.

16. The method of embodiment 11 or 12, wherein the assay is an ELISA.

17. The method of embodiment 16, wherein the ELISA is sandwich ELISA, orcompetitive ELISA.

18. The method of embodiment 17, wherein the competitive ELISA isperformed using an immunoassay plate, wherein the immunoassay plate iscoated with one or more polypeptide consisting essentially of an aminoacid sequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 12.

19. The method of embodiment 16, wherein the antibody or antigen bindingfragment thereof is conjugated to an enzyme.

20. The method of embodiment 19, wherein the enzyme is horseradishperoxidase.

21. The method of embodiment 11 or 12, wherein the antibody or antigenbinding fragment thereof is conjugated to a label.

22. The method of embodiment 21, wherein the label is an enzyme label, aradioisotope, a fluorescent label, or a bioluminescent label.

23. A kit comprising an antibody or antigen binding fragment accordingto any one of embodiments 1-10, said antibody or antigen bindingfragment specifically binding to 3ABC non-structural protein of FMDV,wherein the antibody or antigen binding fragment thereof specificallybinds the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 12.

24. The kit of embodiment 23, wherein said kit further comprising animmunoassay plate coated with a polypeptide comprising the amino acidsequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6 or SEQ ID NO: 12, FMDV non-structural protein 3ABCor combinations thereof.

25. The kit of embodiment 23, wherein the antibody or antigen bindingfragment thereof is conjugated to a label.

26. The kit of embodiment 23, wherein said label is an enzyme label, aradioisotope, a fluorescent label, or a bioluminescent label.

27. The kit of embodiment 23, wherein the antibody is a monoclonalantibody.

28. The kit of embodiment 23, wherein the antibody is a polyclonalantibody.

29. The kit of embodiment 23, wherein the antibody or antigen bindingfragment thereof is a chimeric antibody, a single chain antibody, a scFvantibody, or a Fab fragment.

30. The kit of embodiment 23, wherein the antibody is Mab 40C8 asproduced by hybridoma which is deposited with the American Type CultureCollection with Designation: PTA-122531.

31. A method of producing an antibody comprising immunizing an animalwith an immunogen comprising a polypeptide consisting essentially of SEQID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQID NO: 12, or combinations thereof; and collecting antibodies from saidanimal.

32. The method according to embodiment 31, said method furthercomprising comparing the binding specificity of said collectedantibodies to the monoclonal antibody 40C8.

33. The method of embodiment 31, said method comprising immunizing saidanimal, generating monoclonal antibodies from splenocytes isolated fromsaid animal and comparing the binding specificity of monoclonalantibodies generated by said method with the 40C8 monoclonal antibody.

34. The method according to embodiment 32, wherein said generatedmonoclonal antibody specifically binds the amino acid sequence of SEQ IDNO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 12 as theepitope.

35. The method according to embodiment 32, wherein said generatedmonoclonal antibody specifically binds the amino acid sequence of SEQ IDNO: 4 as the epitope.

36. The method according to embodiment 32, wherein said generatedmonoclonal antibody specifically binds the amino acid sequence of SEQ IDNO: 5 as the epitope.

37. A polypeptide consisting of an amino acid sequence selected from SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12.

38. A polypeptide consisting essentially of an amino acid sequenceselected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 orSEQ ID NO: 12.

39. An immunogenic carrier protein covalently attached to:

a) a polypeptide consisting of an amino acid sequence selected from SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12; or

b) consisting essentially of an amino acid sequence selected from SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12.

40. A composition of matter comprising a polypeptide bound to asubstrate, said polypeptide:

a) consisting of an amino acid sequence selected from SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12; or

b) consisting essentially of an amino acid sequence selected from SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12.

41. A composition of matter comprising a substrate to which an antibodyaccording ant one of embodiments 1-10 is bound.

42. The method of any one of embodiments 11-22, wherein said antibodybinds to the same epitope as the 40C8 antibody.

43. The method of embodiment 42, wherein said antibody is the antibodyproduced by the hybridoma 40C8 or is an antigen binding fragmentthereof.

44. An immunogenic composition comprising an adjuvant and a polypeptide:

a) consisting of an amino acid sequence selected from SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12; or

b) consisting essentially of an amino acid sequence selected from SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12, saidpolypeptide being, optionally, conjugated to an immunogenic carrierprotein.

45. The method of embodiment 11 or 12, wherein the sample is from avaccinated animal, infected animal or a combination of vaccinated andinfected animals.

46. The method of embodiments 11, 12, 17-22 or 45, wherein said methodis a competitive immunoassay and said method comprises:

combining said biological sample with Mab 40C8 or an antigen bindingfragment thereof, or antibodies having the binding specificity of Mab40C8, or antigen binding fragments thereof, prior to contacting saidbiological with one or more 3ABC polypeptide or one or more polypeptidecomprising an epitope comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6 or SEQ ID NO: 12 or a 3ABC polypeptide;

contacting an immunoassay plate coated with one or more 3ABC polypeptideor one or more polypeptide containing an epitope consisting essentiallyof an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12 with an antibody or antigenbinding fragment having the binding specificity of Mab 40C8, optionallywashing said plate and, subsequently contacting said immunoassay platewith said biological sample; or

contacting an immunoassay plate coated with one or more 3ABC polypeptideor one or more polypeptide containing an epitope consisting essentiallyof an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12 with said biological sample,optionally washing said plate and, subsequently contacting saidimmunoassay plate with an antibody or antigen binding fragment havingthe binding specificity of Mab 40C8.

47. The method of embodiment 46, wherein said polypeptide comprises FMDV3ABC.

48. The method of embodiment 46, wherein said method comprisescontacting an immunoassay plate coated with one or more 3ABC polypeptideor one or more polypeptide containing an epitope consisting essentiallyof an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12 with an antibody or antigenbinding fragment having the binding specificity of Mab 40C8, optionallywashing said plate and, subsequently contacting said immunoassay platewith said biological samples, infected animals being identified byreduced binding of antibodies in said biological sample to saidpolypeptide being the inhibition of antibody binding.

49. The method of embodiment 46, wherein said method comprisescontacting an immunoassay plate coated with one or more 3ABC polypeptideor one or more polypeptide containing an epitope consisting essentiallyof an amino acid sequence selected from SEQ ID NO: 3, SEQ ID NO: 4, SEQID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 12 with said biological sample;contacting said immunoassay plate with an antibody or antigen bindingfragment having the binding specificity of Mab 40C8 that is labeled withat least one of a fluorescent label, an enzymatic label or a radiolabel;and detecting the binding of said labeled antibody or antigen.

50. The method of embodiment 49, wherein reduced binding of said labeledantibody indicates that the animal from which the biological sample wasobtained is infected by FMDV.

EXAMPLE MATERIALS AND METHODS Example: Cloning and Expression of 3ABC*His-Tagged Recombinant Protein from E. coli Expression Plasmid

The viral RNA for the FMDV 01 Campos strain was isolated from FMDVinfected BHK-21 cells following manufacturer protocols (RNeasy kit,Qiagen, Valencia, Calif.). The required recombinant protein 3ABC*fragment of the viral genome was transcribed to cDNA using SuperScript™III First-Strand Synthesis System for real-time polymerase chainreaction (RT-PCR) (Invitrogen, Carlsbad, Calif.) following themanufacturer's instructions. The cDNA obtained was then amplified by anoverlap extension PCR (Forward oligonucleotide:5′-CAATTCCTTCCCAAAAATCT-3′ (SEQ ID NO: 7), Reverse oligonucleotide:5′-GTGGTGTGGTTCGGGGTCCAA-3′ (SEQ ID NO: 8). Expected PCR product sizewas 1316 bp). Site-directed mutagenesis and overlapping PCR was used tointroduce a mutation (*) that changes the residue Cysteine at position163 into an Arginine at the active site of the 3C_(pro) viralproteinase.

The schematic representations of the recombinant protein and of theplasmid used for expression in prokaryotic cells are shown in FIG. 8 andFIG. 9. The sense and antisense primers were designed for cloning of3ABC* PCR into pET30c 6×His tag expression plasmid containing a6×His-tag at the N-terminus of the cloned protein. Cloning of the 3ABC*insert was accomplished by BamHI/HindIII restriction endonucleasedigestion using standard molecular biology techniques. Sequence analysisverified the presence of the correct mutant recombinant protein. Thiswas accomplished by using Big Dye Terminator Cycle Sequencing Kits(Applied Biosystems, Foster City, Calif.) and a PRISM 3700 automatedsequencer.

p3ABC* was expressed in E. coli Rosetta (DE3)pLysS competent cells andinduced with addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) asfollows:

Expression:

1. Grow single colony in 5 ml LB broth containing 0.4% glucose and 50μg/ml kanamycin and 20 μg/ml chloramphenicol, overnight at 37° C. and220 rpm in an incubator.

2. Inoculate the starter culture to 50 ml LB broth containing identicalantibiotic concentration.

3. Grow cells at 37° C. and 220 rpm in the incubator to reach OD=0.8.

4. Induce with 1 mM IPTG (final concentration); grow at 37° C. for 4hrs.

5. Freeze the cell pellet in −20° C. freezer.

Solubilization:

1. Thaw the frozen cell pellet on ice for 30 min.

2. Resuspend the cell pellet in BugBuster® reagent (5 ml buffer/g cells)(EMD Millipore, Billerica, Mass.).

3. Add Benzonase (Novagen, Bilerica, Mass.), lysozyme, leupeptin, andpepstatin to 1× concentration.

4. Incubate at room temperature for 20 min with intermittent stirring.

5. Centrifuge at 11,600 rpm in a SL-50T rotor for 20 minutes at 4° C.Discard supernatant.

6. Add same amount of volume (as above) of 1:10 Bugbuster®.

7. Centrifuge at 3000 rpm in a ST-H750 rotor for 20 min at 4° C.

8. Collect the pellet and resuspend in 1:10 concentration of BugBuster®again.

9. Centrifuge at 3000 rpm using ST-H750 rotor for 20 min at 4° C.

10. Resuspend the pellet in 1:10 Bugbuster® and centrifuge at 11,600 rpmfor 20 min at 4° C. Discard the supernatant.

11. The protein was harvested from inclusion bodies and the proteinpellet was solubilized with 100 mM NaH₂PO₄, 10 mM Tris, 8 M Urea pH 8supplemented with 12 mM β-mercaptoethanol (BME) and 10% glycerol for 1hour on ice.

12. The protein solution was centrifuged at 11,600 rpm in a SL-50T rotorfor 20 min at 4° C. The supernatant was collected.

Protein preparations were aliquoted as 100 μl and stored at −20° C. Twolots of 3ABC* were verified in 12% Bis-Tris sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS PAGE) gel (FIG. 10) and can beseen as an approximate 55 kDa band.

A standard BCA protein assay (Thermo Fisher Scientific Inc., Waltham,Mass.) was performed to determine the approximate concentration of thecrude recombinant protein preparation. Table 2 shows the concentrationand inventory of the two lots of recombinant proteins.

TABLE 2 Batch information of recombinant 3ABC* proteins Recombinantprotein Concentration (mg/ml) FMDV 3ABC* Lot 20112A 7.5 FMDV 3ABC* Lot20112B 6.25

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1—Mab 40C8 Production

An embodiment of the current disclosure provides a cELISA that utilizesan indicator monoclonal antibody (Mab), Mab 40C8, in a competitiveformat, i.e. sera from infected animals are used to block the binding ofthe indicator antibody to a target antigen. Mab 40C8 specifically bindsto a B-cell linear epitope NH2-GPYAGPLERQKPLK-COOH (SEQ ID NO: 3) of the3ABC protein of foot-and-mouth disease virus (FMDV) and is a keycomponent of the cELISA. The minimal optimal reactive epitope is mappedto GPLERQ (SEQ ID NO: 4), enabling unique and useful function of the Mabin diagnostic testing that are described here after.

Mab 40C8 was generated using the peptide NH2-CGPYAGPLERQKPLK-COOH, (SEQID NO: 6, FIG. 1, which was chemically synthesized and purified by HPLC.Sequence analysis (n=331 strains available in GenBank), indicates ˜20%genetic variability in this epitope (FIG. 2) indicating that amonoclonal generated against this peptide may recognize these sequencevariants, thus enabling a cross-reactive serological test. Thevariability within the peptide is higher in the amino acid residuesalanine (A), leucine (L), and lysine (K) at position 4, 7 and 11,respectively (FIG. 3).

The peptide was conjugated to KLH and used for mice immunization and Mabproduction using standard protocols. Briefly, female BALB/C mice wereimmunized subcutaneously with 20 μg of the peptideNH2-GPYAGPLERQKPLK-COOH (SEQ ID NO: 3) conjugated to KLH emulsified inan equal volume of Complete Freund's Adjuvant. Three identical boostersemulsified in incomplete Freund's adjuvant were given at four weeksinterval. The mice were boosted with the peptide-KLH conjugate in PBS(40 μg/mouse) by intraperitoneal injection 4 days before fusion.Immunized spleen cells were fused with myeloma cells (P3X63 Ag8.653).After 2 weeks, hybridoma supernatants were screened using the FMDVrecombinant 3ABC as the antigen in an indirect ELISA. Mab 40C8 exhibitedstrong reactivity with the 3ABC antigen and was characterized as IgG1isotype with kappa light chains.

Mab 40C8 Reactivity to Peptides and 3ABC Recombinant Protein

Additional indirect ELISA testing confirmed reactivity to diverse 3ABCpeptide variants (FIG. 4). This result indicates that this Mab exhibitsbroad peptide reactivity, potentially enabling detection of diversestrains of FMDV (n=331 by in silico analysis, as depicted in FIG. 2).The minimal epitopes are GPLERQ (SEQ ID NO: 4), and residues 2 and 3 ofthis sequence are not critical for Mab 40C8 binding.

Mab 40C8 also exhibited strong Western blot reactivity to the 3ABCrecombinant protein, consisting of all sequence variants within the 3ABCepitope (FIG. 5). Furthermore, this Mab was able to compete with serafrom a FMDV infected animals to the recombinant 3ABC protein,demonstrating the feasibility of a competitive ELISA application.

Mab 40C8 Reactivity to the Six FMDV Serotypes Infected Cell Lysates

For a cELISA, Mab 40C8 was directly conjugated to the horseradishperoxidase (HRP) detection system and the reactivity of Mab 40C8conjugated to HRP (40C8-HRP) was screened with a panel of fifteen FMDVisolates using a direct ELISA format. These FMDV antigens were preparedfrom a cell line known to be highly permissive to FMDV growth (LFBKαvβ6). FIG. 6 shows that in two separate experiments 40C8-HRP bound toFMDV antigen in infected cell lysates. Variability in binding (e.g.,measured by 450 nm absorbance) can be attributed to differences in theFMDV antigen content in the cell lysates. For example, isolates such asserotype O Israel (O Isr), A24 Cruzeiro SDG (A24 SDG), and SAT-2 thatdid not bind efficiently to Mab 40C8 in this assay were suspected of notgrowing well since other isolates of the same serotype and strain werereactive to Mab 40C8 using Western Blot analysis (see FIG. 7 andassociated result descriptions below). Moreover, a similar lowimmunoreactivity for O Isr and A24 SDG was observed in anotherexperiment (data not shown) in which the pan-FMDV specific monoclonalantibody, F1412SA (F14), which specifically binds an epitope in the FMDVVP2 (1B) protein, was used as a positive control. Collectively, theseELISA results indicate that Mab 40C8 exhibited reactivity to six FMDVserotypes using cell-lysates. SAT-1, the seventh serotype, was notavailable at the time of this analysis.

Furthermore, Western blot analysis was also performed to verify theELISA results. Cell lysates prepared from BHK-21 cells infected with FMDviruses representing different serotypes were mixed with 2× Laemmlibuffer, boiled, and stored. For Western blot analysis, a 1/10 fractionof cell extracts was run on 12% SDS-PAGE gels and transferred ontonitrocellulose blots following standard procedures. The blots wereprobed using Mab 40C8. The results are shown in FIGS. 7A and 7B. As acontrol, and to demonstrate that all samples contained the viralantigen, a second Western blot was developed using a FMDV 3D-specificmonoclonal antibody (see upper panel in FIG. 5A where a product of about52 KDa is detected that corresponds to the FMDV protein 3D). Togetherthese results show that Mab 40C8 reacted with FMDV 3ABC across sixstrains in four serotypes. Certain strains exhibit strong reactivitywhile others required higher concentration of the Mab 40C8. Thecollective ELISA and Western blot data demonstrate that Mab 40C8specifically binds FMDV proteins from all available serotypes.

SAT-2 Reactivity Confirmation

In the initial studies, characterizing the reactivity of the Mab 40C8with cell culture-grown FMDV serotype SAT-2, results indicated that onestrain of SAT-2 was not reactive, as seen in FIG. 6, and FIG. 7B.However, when another SAT-2 strain was used, there was a positivereaction (FIG. 7A). In additional studies using different strains ofSAT-2 in which animals were infected, all three serum samples werepositive. So, it appears that the one strain of SAT-2 was not reactivedue to low cell culture growth.

Mab 40C8 Non Reactivity to Ad-FMD Vaccines Infected Cells

The current disclosure provides a cELISA that can also discriminatebetween animals infected with FMDV and those immunized with FMD vaccinesthat have been inactivated and purified of NSPs. The FMD vaccineimmunized animals (immunized using inactivated, purified FMDV vaccine oran adenovector serotype 5 vaccine) do not contain FMDV protein 3ABCwhich is recognized by Mab 40C8-HRP. For example, a vaccine containingthe molecular clone of the adenovector serotype 5 carrying the FMDVcapsid and processing genes (Ad-FMD) lack the specific 3ABC immunogenicsite in the FMDV 3ABC protein. The antibody responses of animalsimmunized with vaccines lacking the viral non-structural proteins (e.g.Ad-FMD or inactivated, purified FMDV vaccines) can be discriminated ordifferentiated from those responses exhibited by FMDV infected animals.These differences can be detected by companion diagnostic tests,so-called DIVA (differentiation between infected and vaccinatedanimals). The Ad-FMD vaccines were designed to be vaccines with DIVAcapabilities using a FMDV 3ABC cELISA designed as a companion diagnosticassay. The reactivity of Mab 40C8 with Ad-FMD infected cells wasscreened with FMDV Asia 1, O1 Campos, and O1 Manisa constructs using adirect ELISA as above. Ad-FMD-infected M2A cells actively express FMDVcapsid and some processing genes. Ad-FMD antigens (infected M2A celllysates) were directly adsorbed to ELISA plates and processed as above.Table 3 shows the clear binding of Mab F14 to the expressed FMDV capsidproteins, and the lack of binding of Mab 40C8 to the Ad-FMD cell lysatesthat lack immunogenic FMDV 3ABC nonstructural proteins. Therefore, theELISA of the present disclosure can serve as a companion diagnosticassay for Ad-FMD vaccines.

TABLE 3 Immunoreactivity of Mab 40C8 and Mab F14 to Ad-FMD vaccineconstructs grown in M2A cells. Highest dilution for a Adenovector FMDvaccine positive response construct Mab 40C8 Mab F14 Ad5-ASaudi Arabia95  <1:2* >1:64 Ad5-O1 Manisa <1:2 >1:64 Ad5-O1 Campos <1:2 >1:64Ad5-O1Campos.2B <1:2 >1:64 Ad5-O1Campos.2B + F (RGD) <1:2 >1:64 M2Acells (no vaccine control) <1:2 <1:2  *No positive reaction detected atthe lowest dilution tested (1:2)

Example 2—3ABC cELISA Kit

Preparation of Horse-Radish Peroxidase (HRPO)-Conjugated Mab 40C8

Mab 40C8 ascites purified by sodium ammonium sulfate cut method werediluted in PBS to provide 2 mg/ml antibody. Mab 40C8 was dialyzedagainst 0.1 M sodium bicarbonate/carbonate buffer, pH 9.6. Half ml of a4 mg/ml horseradish peroxidase solution (HRPO) dissolved in distilledwater was mixed with 100 μl of freshly prepared 0.1 M sodium periodateby stirring for 20 minutes at room temperature. The HRPO/sodiumperiodate solution was dialyzed against 1.0 mM sodium acetate buffer, pH4.4 at 4° C. The pH of HRPO/sodium periodate solution was adjusted from4.05 to pH 9.6 by adding 15 μl of freshly prepared 0.1 M sodiumcarbonate. The dialyzed Mab were combined with the HRPO/sodium periodatesolution, and stirred for 2 hours at room temperature. Fifty μl offreshly prepared 4 mg/ml sodium borohydride solution was slowly added tothe HRPO/FMDV ascites SAS Cut solution, incubated for 2 hours at 4° C.,and then dialyzed against 1×PBS. The HRPO-conjugated Mab 40C8 wasstabilized by adding a final concentration of 10% heat inactivated goatserum, 0.01% thimerosal, and 0.03% WAWA (4-aminoantipyrine).

Example: Preparation of Recombinant 3ABC-Coated Immunoassay Plate

A dilution of recombinant 3ABC was made in 0.1 M carbonate/bicarbonatebuffer. Immunoassay plates were filled with 50 μl antigen per well,incubated in humid chamber overnight at 4° C. Wells were blocked with 20μl blocking agent by incubating for 2 hours at 37° C. in humid chamber.The plates were dried overnight after discarding the liquid.

Example: Optimizations of Other Format Variables and Formulation of KitComponents

Other format variables including serum dilution factor, serum incubationtime, buffer types for serum dilution, conjugate dilution buffer andplate wash were thoroughly compared and the combination supporting bestanalytical sensitivity and specificity was chosen. Components of the kitwere formulated as follows.

1. Test serum dilution: PBS

2. Serum dilution factor: 1:2

3. Conjugate diluting buffer: 1:2 dilution of Stabilzyme (SurModics,Inc., Eden Prairie, Minn.) in PBS

4. Wash buffer: PBS containing 0.1% TWEEN (polysorbate 20)

5. Color reaction substrate: TMB (3,3′,5,5′-Tetramethylbenzidine)

Preparation of Positive and Negative Controls

FMDV antibody negative bovine and porcine from FMDV-free farms in theU.S. were screened using a 3ABC cELISA. One bovine and three porcinewere first immunized with recombinant 3ABC emulsified in CompleteFreund's Adjuvant and then boosted with the same antigen emulsified inIncomplete Freund's Adjuvant. Serum from a FMDV negative bovine was usedas the negative control in the assay. Positive bovine and porcinecontrol serum was also collected and used to validate the immunoassay.These non-infectious positive controls showed reliable andconcentration-dependent positive results in the cELISA format (Table 4).

Example: Assay Procedure

1. Add 50 μl serum at 1:2 in PBS to each well and incubate 30 min atroom temperature.

2. Wash plate three times with 250 μl per well.

3. Add 50 μl HRP conjugated Mab and incubate at room temperature for 30min.

4. Wash plate 3 times with 250 μl per well wash buffer.

5. Add 50 μl substrate per well and incubate 20 min at room temperature.

6. Add 50 μl Stop solution to each well and read at 450 nm.

TABLE 4 Representative QC data tested with sensitivity panel and controlsera (bovine positive and negative) % Sample I.D., Dilution OD OD MeanInhibition C673 (+), 1:20 0.204 0.198 0.201 77.4 C673 (+), 1:40 0.2740.278 0.276 68.9 C673 (+), 1:80 0.392 0.393 0.393 55.8 C673 (+), 1:1600.602 0.598 0.600 32.5 Kit (−) 0.905 0.924 0.889 0.0 0.849 0.876 Kit (+)0.265 0.266 0.259 70.8 0.250 0.256

Example: Analytical Sensitivity Evaluation

Seven FMDV Serotypes Limit of Detection Analysis

In order to determine the analytical sensitivity of the assay, seracollected from animals infected with one of each of the seven FMDVserotypes were diluted two-fold, to a maximum dilution of 1:2048.Samples from each dilution were evaluated in the 3ABC ELISA twice andonce in a commercially available assay according to manufacturer'sinstructions (PrioCHECK® FMDV NS Antibody ELISA, ThermoFisherScientific, Grand Island, N.Y.). The highest dilution at which apositive reaction was recorded is listed in Table 5. The analyticalsensitivity for the 3ABC ELISA was at least 16- to 32-fold moresensitive for the Asia-1 serotype sample (cut-off dependent), two-foldmore sensitive for serotype O 1, comparable sensitivity for serotypes A,C, SAT-1, and SAT-2, and one dilution less sensitive for SAT-3 comparedto the PrioCHECK® FMDV NS Antibody ELISA. Sera collected from animalsinfected with one of the seven FMDV serotypes were reactive in bothassays, and the sensitivity for the 3ABC ELISA was optimal for acompetitive ELISA format.

TABLE 5 Analytical Sensitivity of the 3ABC ELISA for seven FMDVserotypes with 35% inhibition as the cut-off (1st test) (45% inhibitionas the cut-off in the 2nd test). For example, 1:2048 detection isbetter/more sensitive than 1:32. Highest dilution for a positiveresponse 3ABC ELISA 3ABC ELISA FMDV serotype (1^(st) test) (2nd test)PrioCHECK ® A 1:512 (256) 1:256 (128)  1:128 Asia-1 1:2048 (512) 1:2048(1024) 1:32 C 1:64 (32) 1:128 (32) 1:32 O 1 1:512 (256) 1:256 (128) 1:64SAT-1 1:512 (256) 1:256 (128)  1:128 SAT-2 1:128 (64) 1:64 (64) 1:32SAT-3 1:32 (16) 1:32 (16) 1:32

Post-Infection Antibody Detection Time Point/Window Determination

One male castrated steer was infected intradermolingually (IDL) with oneFMDV serotype, (4 calves were each infected with one of 4 FMDVserotypes, A Iraq 2009, Asia 1 Shamir, SAT-1, and O Israel 2008). Foreach IDL-challenge calf, four uninfected cattle were mingled in order tobe infected via the contact route. Serum samples were collected fromeach animal nearly every day from days 0 to 14, and then on days 21 or22, 28, and 35 or 36.

The 3ABC ELISA detected antibodies in sera from cattle infected with oneof the four FMDV serotypes between 7 to 10 days post-infection (dpi)(FIG. 11). Antibodies to FMDV O Israel 2008 were detected by 7 dpi, toAsia 1 Shamir by 8 dpi, to A Iraq 2009 by 9 dpi, and to SAT-1 by 10 dpi.

In another embodiment designed to determine when antibodies to the FMDV3ABC nonstructural proteins can be detected by the 3ABC ELISA followinginfection, sera were collected over time from cattle that were infectedwith one of three FMDV serotypes, SAT-1, SAT-2, or SAT-3. Sera fromvarious days post-infection (dpi) were evaluated using the 3ABC ELISA.The first day on which there was a positive reaction in the 3ABC ELISAare summarized in Table 6.

TABLE 6 Detection of antibodies to FMDV nonstructural proteins fromFMDV- infected cattle. Days Post-Infection when antibodies to FMDVnon-structural proteins were detected in cattle Test used to detect serafollowing infection with one of three antibodies to FMDV FMDV serotypes(no. cattle) non-structural proteins SAT-1 (n = 2) SAT-2 (n = 2) SAT-3(n = 2) 3ABC ELISA 7, 10 6, 4 10, 11

These data indicates that the new FMDV 3ABC ELISA will be useful indetecting antibodies in cattle within 7 to 13 days post-infection withany one of the FMDV serotype SAT 1, SAT-2, or SAT-3 strains.

Example: Analytical Specificity Evaluation

FMD Look-Alike Samples Specificity Analysis

Diagnostic bovine sera samples previously evaluated and identified ashaving been collected from animals infected with pathogens that causeFMD look-alike lesions were tested with the 3ABC ELISA, the FMDV 3Dvirus infection associated antigen agar gel immunodiffusion (VIAA AGID),and by assays specific for the pathogen, e.g. Vesicular Stomatitis Virus(VSV) ELISA. Data are summarized in Table 7.

TABLE 7 Specificity of FMDV 3ABC ELISA on diagnostic serum samples thatwere confirmed as negative for FMDV antibodies and positive for VSVantibodies. No. of No. Identified as Positive samples FMDV 3D FMDV 3ABCtested VSV ELISA VIAA AGID ELISA Specificity 55 55 0 0 100%

The specificity was 100% for sera tested from 55 cattle that had beeninfected with VSV and were positive for VSV antibodies. In the 3ABCELISA, all samples exhibited ≤28% inhibition with an average of −12%inhibition and a standard deviation of 25%.

In another experiment, 44 samples from 8 species of FMDV-susceptibleanimals that exhibited vesicular lesions but were free of FMDV, weretested by three assays: 1) FMDV 3ABC ELISA, 2) FMDV 3D VIAA AGID, and 3)FMDV PrioCHECK. Data are summarized in Table 8.

TABLE 8 Specificity of FMDV 3ABC ELISA on sera from vesicular diseasediagnostic samples that were confirmed as FMDV negative No. IdentifiedNo. Identified as Negative As Positive Animal FMDV 3ABC FMDV 3D FMDV byany of Species ELISA VIAA AGID PrioCHECK ® the three assays Alpaca 2 2 20 Bison 1 1 1 0 Bovine 28 28 28 0 Buffalo 1 1 1 0 Goat 4 4 4 0 Ovine 2 22 0 Swine 4 4 4 0 Yak 2 2 2 0 Total 44 44 44 0

The specificity of the 3ABC ELISA was 100% for 44 samples collected from8 species of animals that were FMDV free and exhibited vesicularlesions, i.e. rule-out data provided by the USDA Animal and Plant HealthInspection Service Foreign Animal Disease Diagnostic Laboratory. Thesame results were obtained in the FMDV PrioCHECK® FMDV NS Antibody ELISAand the FMDV 3D VIAA AGID assays.

Adenovirus-A24 FMD Vaccine Vaccinated Samples Specificity Analysis

Cattle from Michigan and Nebraska were vaccinated with the conditionallylicensed Ad-A24 FMD vaccine (USDA product code 1FM1.R0). Forty-ninecattle serum samples collected approximately 11 months post-vaccinationwere positive using serum virus neutralization (SVN) to detectantibodies produced to the FMDV A24 capsid proteins (average titer=1.5log 10, std. dev.=0.5, range 0.9 to 2.7 log 10) (FIG. 12). Antibodieswere not detected to the FMDV non-structural proteins, as expected,since these animals had been vaccinated but not infected with FMDV. Inthe 3ABC ELISA, the average % inhibition was 1.5%, std. dev.=16%, andrange=−29% to 31%. There was no correlation between the SVN titers tothe A24 antigen and the percent inhibition measured in the 3ABC ELISA;correlation=0.025 (R²=0.0006). This data indicates that the 3ABC ELISAis suitable as a companion diagnostic assay for AdFMD based vaccines.

Example: Diagnostic Sensitivity Evaluation

Positive Reference Sample Set (Known Positive Status) Analysis

Serum samples obtained from 139 animals, at least 10 to 14 dayspost-infection with a known serotype of FMDV, were evaluated for theirresponse in the 3ABC ELISA and with the commercially availablePrioCHECK® FMDV NS Antibody ELISA. The samples were obtained from 112cattle, 18 pigs, 5 sheep, and 4 goats that were experimentally infectedwith one of the seven FMDV serotypes; this sample set covered the fullspectrum of all seven serotypes. All 139 serum samples were positivewith the FMDV 3ABC ELISA assay, and 136 of those samples were positivewith the PrioCHECK® FMDV NS Antibody ELISA (Table 9). The resultsdemonstrate that the 3ABC ELISA can detect antibodies produced to theseven serotypes of FMDV 3ABC nonstructural protein in diverse animalspecies.

The diagnostic sensitivity was calculated using the results from the 139serum samples (Table 10). Since all 139 serum samples that werecollected from FMDV-infected animals (known positives) exhibited apositive response in the 3ABC ELISA, the diagnostic sensitivity was100%. In the PrioCHECK® FMDV NS Antibody ELISA, 136 of the same 139samples were positive, which indicates a diagnostic sensitivity of97.8%. The % inhibition (% I) distribution of these samples in the 3ABCELISA are provided graphically on the right side of the vertical line inFIG. 13.

TABLE 9 Detection of antibodies to FMDV produced in animals infectedwith one of seven serotypes of FMDV FMDV Serotype* 3ABC ELISA PositiveFMDV PrioCHECK ® Positive A 52 52 O 23 23 C 1 1 SAT1 22 20 SAT2 3 3 SAT320 20 Asia-1 18 17 Total 139 136 *Serum samples were from 112 bovine, 18swine, 5 ovine, and 4 caprine experimentally infected animals.

TABLE 10 Diagnostic sensitivity of the 3ABC ELISA compared to thePrioCHECK with reference to samples that were obtained from animalsinfected with FMDV. FMDV Assay Result 3ABC ELISA PrioCHECK Positive 139136 Negative 0 3 Total Samples Tested (Known Positives) 139 139Diagnostic Sensitivity 100% 97.8%

Diagnostic Specificity Evaluation

Approximately 500 bovine sera were split into two sample sets and testedby two laboratories, to assessed diagnostic specificity. At TVMDL, therewere 4 false positives out of 491 serum samples using 45% inhibitioncut-off, resulting in 99.2% diagnostic specificity (FIG. 14). At FADDL,one false positive (out of 486) was obtained, resulting in 99.8%diagnostic specificity; this one false positive was one of the fourfalse positives identified at TVMDL, i.e., same biological sample.Comparison of the TVMDL and FADDL % inhibition for the samples resultedin TVMDL % I average (Avg)=−0.018, standard deviation (SD)=14.6; FADDL %I Avg=−7.48, SD=22.5; p value <0.05. Although a significant differencein % I was observed, equivalent diagnostic specificity (probability thata negative sample test negative) was obtained; TVMDL Dx sp=99.2% andFADDL Dx sp=99.8%. This indicates that the assay of the currentdisclosure exhibits a wide % I distribution range for negatives (seeFIGS. 12, 13, and 14) enabling tolerance of % I differences between labsand testers and high diagnostic specificity.

Furthermore, 200 samples from this 491 sample set was also tested withthe PrioCHECK® FMDV NS Antibody ELISA at FADDL and 8 false positiveswere identified, resulting in 96% diagnostic specificity; only one ofthe false positives corresponded to the 3ABC ELISA false positiveidentified by TVMDL, i.e., same biological sample.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

We claim:
 1. An isolated monoclonal antibody or antigen binding fragmentthereof that specifically binds to 3ABC non-structural protein ofFoot-and-Mouth Disease virus (FMDV), wherein the antibody or antigenbinding fragment thereof specifically binds an epitope consistingessentially of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 orSEQ ID NO:
 12. 2. The antibody or antigen binding fragment thereof ofclaim 1 that specifically binds the amino acid sequence of SEQ ID NO: 3,SEQ ID NO: 6, or SEQ ID NO: 12 as the epitope.
 3. The antibody orantigen binding fragment thereof of claim 1 that specifically binds theamino acid sequence of SEQ ID NO: 4 as the epitope.
 4. The antibody orantigen binding fragment thereof of claim 1 that specifically binds theamino acid sequence of SEQ ID NO: 5 as the epitope.
 5. The antibody orantigen binding fragment thereof of claim 1, wherein the antibody isselected from a chimeric antibody, a single chain antibody, a singlechain fragment variable (scFv) antibody, or a fragment antigen-binding(Fab fragment).
 6. The antibody or antigen binding fragment thereof ofclaim 1, wherein the antibody or antigen binding fragment thereof isconjugated to a label.
 7. A method of detecting FMDV infection in ananimal, the method comprising contacting a sample from an animal with amonoclonal antibody according to claim
 1. 8. The method of claim 7,wherein the sample obtained from the animal is a body-fluid sample or atissue sample.
 9. The method of claim 8, wherein the body-fluid sampleis aqueous humor, vitreous humor, blood serum, blood plasma,cerebrospinal fluid, endolymph, perilymph, exudates, lymph, mucus,pericardial fluid, pleural fluid, synovial fluid, milk, or oral fluids.10. The method of claim 8, wherein the tissue sample is brain, eyes,pineal gland, pituitary gland, thyroid gland, parathyroid glands,thorax, heart, lungs, esophagus, thymus gland, pleura, adrenal glands,appendix, gall bladder, urinary bladder, large intestine, smallintestine, kidneys, liver, pancreas, spleen, stoma, prostate gland,testes, ovaries, or uterus.
 11. A kit comprising a monoclonal antibodyor antigen binding fragment according to claim 1, said antibody orantigen binding fragment specifically binding to 3ABC non-structuralprotein of FMDV, wherein the antibody or antigen binding fragmentthereof specifically binds the amino acid sequence of SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 12.12. The kit of claim 11, wherein said kit further comprising animmunoassay plate coated with a polypeptide comprising the amino acidsequence selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6 or SEQ ID NO: 12, FMDV non-structural protein 3ABCor combinations thereof.
 13. The kit of claim 11, wherein the antibodyor antigen binding fragment thereof is conjugated to a label.
 14. Amethod of producing an antibody comprising immunizing an animal with animmunogen comprising a polypeptide consisting essentially of SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO:12, or combinations thereof; and collecting antibodies from said animal.15. A polypeptide consisting of or consisting essentially of an aminoacid sequence selected from SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6 or SEQ ID NO:
 12. 16. An immunogenic carrier proteincovalently attached to a polypeptide according to claim
 15. 17. Acomposition of matter comprising a polypeptide according to claim 15bound to a substrate.
 18. A composition of matter comprising a substrateto which an antibody according to claim 1 is bound.